Novel biomarker for monitoring neuronal death and Alzheimer’s disease progression

More than a century ago, Alois Alzheimer described an “unusual disease of the cerebral cortex” which progressively caused memory loss, disorientation, hallucination and ultimately, death. He noticed a thinning of the cerebral cortex and identified the presence of amyloid plaques along with neurofibrillary tangles. In the mid-eighties, scientists established that these plaques and tangles were composed respectively of the 42-amino acid β-amyloid protein (Aβ42) and the microtubule-associated protein Tau (D.M. Holtzman et al., 2011). Accumulation of amyloid plaques within the brain is now viewed as toxic to synapses and neurons as it results in the binding of harmful metals, the production of reactive oxygen species and the direct damage to cellular membranes.

In 2004, the launch of a radio-labeled analog of the amyloid-binding dye Thioflavin T [Pittsburgh compound B (PiB)] capable of penetrating the blood-brain barrier enabled researchers to capture images of fibrillar amyloid deposits in vivo and have a greater understanding of Alzheimer’s (AD) pathogenesis. Unfortunately, there is still an urgent need for novel biomarkers with diagnostic and drug screening capabilities. Recent studies suggested that the Aβ peptide is a multifunctional peptide with non-pathological effects and that its association with AD is done in conjunction with the expression of amyloid precursor proteins (APP). Combination of these two factors results in the imbalance between memory formation and memory loss (H.S. Nhan et al., 2014).

The primary function of APP is yet to be described but they seem to be implicated in the regulation of the formation of synapses. There are three major APP isoforms (APP695, APP751 and APP770) formed through alternative splicing of precursor mRNA. They are transmembrane proteins best known to be the precursor molecule for β-amyloid proteins (Aβ40, Aβ42 and Aβ43 being the most common forms). The APP695 isoform is preferentially expressed in the central nervous system, while APP751 and APP770 are highly expressed in peripheral tissues (H.S. Nhan et al., 2014). It has been demonstrated that APP695 can be cleaved by caspases at an intracellular site (Asp644) resulting in the release of two pro-apoptotic entities with potential involvement in the etiology of the disease: a 31-amino acid C-terminal peptide (C31) and a larger neo-APP fragment termed APP ΔC31 (E. Bertrand et al., 2001; V. Galvan et al., 2002). Recent studies highlighted the importance of the cleavage event in the comprehension of the progression of AD (J.A. Harris et al., 2010; S.S. Park et al., 2012). Histological analysis of human brain tissue also demonstrated that this cytoplasmic cleavage is four-fold greater in patients with AD than in control samples and that the products of this cleavage are mainly localized to plaques and tangles in key areas of the brain affected by the disease.

The APP ΔC31 truncated protein was shown to accumulate in brain tissue of AD mouse models and AD patients. Consequently, the specific measurement of APP ΔC31 when combined with measurements of other AD-associated proteins such as Aβ40/42, sAPPα and tau/p-tau could prove to be a useful biomarker for the diagnosis and monitoring of AD progression. To enable the quantitative measurement of APP ΔC31 formation in cells and tissue, scientists at Enzo Life Sciences collaborated with the Buck Institute for Research on Aging (USA) and developed a colorimetric ELISA kit. Currently, researchers detect APP ΔC31 by Western blot, which is time-consuming, laborious, less sensitive, semi-quantitative and very often inconsistent. ELISA comes as a very sensitive and reliable alternative and its utility in a drug screening application was demonstrated by Descamps et al. (2011) in APP770-transfected 7W CHO cells where treatment with Simvastatin led to the generation of APP ΔC31 via stimulation of intracellular caspase cleavage.

A fully quantitative APP ΔC31 ELISA kit, which enables the measurement of an important cleavage fragment associated with AD offers great promise for screening inducers or inhibitors of caspase-mediated cleavage of APP695 at Asp644. A worldwide research effort during the past quarter century has yielded an increasingly detailed picture of the cytopathological, biochemical, and genetic underpinnings of the disease. It is hoped that biomarkers of quality such as APP ΔC31 will attract further studies for validation and represent new target for AD diagnostic and therapeutic development.